This experiment explored the mechanism underlying benzo[a]pyrene (BaP) distribution in different subcellular fractions of the earthworm Eisenia fetida (Fraction C: associated with the cytosol; Fractions D: associated with granules; and Fraction E: associated with tissue fragments and cell membranes) under conditions of contamination with both BaP and cadmium ions (Cd²⁺) at different concentrations. The results showed that Cd²⁺ inhibited the accumulation of BaP in the earthworm, and that BaP was accumulated to the greatest extent in Fraction E, followed by Fraction C and Fraction D. With the addition of Cd²⁺, BaP concentrations in the three fractions initially decreased, but subsequently increased with the increasing concentration of Cd²⁺, whereas protein content and acetylcholinesterase (AChE) activity showed the opposite trend. In contrast, glutathione s-transferase (GST) activity initially decreased and then subsequently increased with increasing Cd²⁺ concentration in Fraction C and E, whereas the activity gradually increased in Fraction D. Correlation analysis indicated that protein content showed a significant negative correlation with BaP concentration in Fraction C and E; AChE activity showed a significant negative correlation with BaP concentration in Fraction C; and GST activity showed a non-significant correlation with BaP concentration. Collectively, the results indicate that BaP mainly accumulated in Fraction E, and that the accumulation of BaP in Fraction C and E was correlated with the content of protein and activity of AChE, which was influenced by Cd²⁺. Furthermore, BaP concentration initially decreased, but subsequently increased, with the increasing concentration of Cd²⁺.